Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displaying in vitro efficacy against melanoma cells

Abstract

CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5-9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 μM. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds.

Keywords: CDC25 phosphatases; cancer; cell cycle; drug discovery; melanoma cells.

Figure 1. Known quinone-containing CDC25 Inhibitors

Figure 2. Flow chart of the multiple ligand-based chemoinformatic strategy implemented in this work

Figure 3. Effect of compound 7 on the Lineweaver-Burk plots of the A. CDC25A B. CDC25B and C. CDC25C phosphatase activity

The phosphatase activity was measured through the rate of OMFP hydrolysis as described in the Materials and Methods. The activity was determined either in the absence (empty circles in A., B. and C.) or in the presence of the following concentrations of compound 7: 0.5 μM (filled triangles in A.), 0.25 μM or 0.5 μM (filled triangles or filled circles, respectively, in B.), 1 μM (filled triangles in C.).

Figure 4. Effect of NSC 119915 and its close analogs on cell growth rate of melanoma cells

A. A2058 and B. SAN cells were incubated for 48 h with 0.5% DMSO as a vehicle control or 100 μM of each of the indicated compounds. The cell growth rate is reported as arbitrary units (a.u.). Data from four independent experiments are reported as the means ± SE. *p < 0.05 and **p < 0.01 compared to control cells.

Figure 5. Effect of compound 7 on the distribution of cell cycle phases of A2058 cells

The determination of cells in the different phases was evaluated after A. 16 h or B. 24 h from treatment with 0.5% DMSO or 100 μM compound 7, as described in the Materials and Methods. Histograms, which show the cell percentage among the various phases, were obtained from triplicate experiments and reported as the means ± SE. *p < 0.05 and **p < 0.01 compared to control cells. Vehicle alone, open bars; compound 7, black bars.

Figure 6. Effect of compound 7 on CDC25A, B and C protein levels

Total protein extracts from A2058 cells, incubated with 0.5% DMSO (open bars) or 100 μM compound 7 (black bars) for 2, 4, 8 or 16 h, were analyzed by Western blotting. GAPDH was used as loading control. The doublets observed in the immunoblots detecting CDC25A or CDC25C were due to the common presence of multiple isoforms of these proteins. Densitometric analysis is shown in the lower panel. Data from triplicate experiments were reported as the means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, compared to control cells. Other details as described in the Materials and Methods.

Figure 7. Effect of compound 7 on the apoptosis of A2058 and SAN cells

The apoptotic process was evaluated in A2058 (panels A. and C.) and SAN (panels B. and D.) cells either through the determination of the number of cells with a subdiploid DNA content (A. and B.) or by measuring the caspase-3 enzymatic activity (C. and D.). Cells were treated with 0.5% DMSO (open bars) or 100 μM compound 7 (black bars) for the indicated incubation times. Apoptosis was expressed as a percentage, whereas caspase-3 activity was reported as arbitrary units (a.u.). Data from triplicate experiments were reported as the means ± SE. *p < 0.05, **p < 0.01, ****p < 0.0001, compared to control cells. Other details as described in the Materials and Methods.

Figure 8. Effect of Z-VAD-FMK, a pan-caspase inhibitor, on the apoptotic process induced in melanoma cells by compound 7

A2058 (panel A.) and SAN (panel B.) cells were treated with 0.5% DMSO or 100 μM compound 7 and incubated in the absence or in the presence of 100 μM Z-VAD-FMK; the apoptosis was evaluated after a 48-h incubation through the determination of the number of cells with a subdiploid DNA content. Data from triplicate experiments were reported as the means ± SE. **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to respective control cells. Other details as described in Materials and Methods.

Figure 9. ROS production and their involvement in the apoptotic process of A2058 cells, as induced by treatment with compound 7

A. Time-dependent measurement of ROS production. Cells were incubated with 0.5% DMSO (open bars) or 100 μM compound 7 (black bars) and then the intracellular ROS level was measured. B. Effect of antioxidant molecules on ROS production. The ROS level was also measured in cells untreated or pretreated with NAC or apocynin after a 4-h incubation with DMSO or 7. C. Effect of NAC on apoptosis. The PI incorporation was evaluated in cells untreated or pretreated with NAC after a 48-h incubation with DMSO or 7. ROS production was expressed as a.u., and apoptosis as a percentage. Data from triplicate experiments were reported as the means ± SE. *p < 0.05 and **p < 0.01 compared to control cells. Other details as described in the Materials and Methods.

Figure 10. Effect of compound 7 on some apoptotic mitochondrial markers in A2058 cells

A. Measurement of the mitochondrial membrane depolarization. Cells were treated for the indicated times with 0.5% DMSO (open bars) or 100 μM compound 7 (black bars). B. Evaluation of Bcl-2 and Bax protein levels. Total protein extracts from A2058 cells, incubated with 0.5% DMSO or 100 μM compound 7 for 8 or 16 h, were analyzed by Western blotting. β-actin was used as loading control. C. Densitometric analysis of the Bcl-2 and Bax protein levels, as well as of the Bcl-2/Bax ratio. D. Determination of the caspase-9 enzymatic activity. Total protein extracts from A2058 cells, incubated with 0.5% DMSO (open bars) or 100 μM compound 7 (black bars) for 24 h, were assayed for caspase-9 activity. Data from triplicate experiments were reported as the means ± SE. *p < 0.05, **p < 0.01, ****p < 0.0001, compared to control cells. Other details as described in the Materials and Methods.

Figure 11. Effect of compound 7 on pAkt and p53 protein levels

A. Evaluation of pAkt protein levels. Total protein extracts from A2058 cells, incubated with 0.5% DMSO or 100 μM compound 7 for 2 or 4 h, were analyzed by Western blotting using an antibody raised against pAkt (Ser473). Akt was used as loading control. Densitometric analysis shown in the lower panel. B. Evaluation of p53 protein levels. Total protein extracts from A2058 cells, incubated with 0.5% DMSO or 100 μM compound 7 for 8 or 16 h, were analyzed by Western blotting. GAPDH was used as loading control. Densitometric analysis shown in the lower panel. Data from triplicate experiments were reported as the means ± SE. *p < 0.05, ***p < 0.001, compared to control cells. Other details as described in the Materials and Methods.